Abstract
Introduction Diagnosis of very short-segment Hirschsprung's disease (vsHD) by rectal suction biopsy
is challenging as its aganglionic zone (AZ) overlaps with physiologic hypoganglionic
zone and calretinin-positive mucosal nerves may extend from the transition zone (TZ)
into AZ. We studied whether an increasing trend/gradient of calretinin-positive mucosal
nerves along the distance from AZ toward TZ aids in diagnosis of HD.
Materials and Methods In this study, 46 rectal suction biopsies from non-HD and HD, and 15 pull-through
specimens from short-segment HD were evaluated by mucosal calretinin immunostain (CI)
and image processing and analysis (IPA) to measure pixel count (PC, the percentage
of calretinin stained pixels in the mucosa). Consecutive longitudinal sections of
proximal AZ toward distal TZ in HD pull-through specimens were utilized as a vsHD
surrogate model. First, we studied variability of mucosal CI in non-HD biopsies along
the distance from dentate line. Second, we determined a cutoff point of mucosal CI
by IPA that separated non-HD versus HD and applied this cutoff to longitudinal sections
from proximal AZ to distal TZ segments in HD pull-through specimens. Third, we studied
whether an increasing trend of mucosal CI was universally observed in HD pull-through.
Results Our findings included a significant variability in PC along the biopsy distance in
non-HD cases. Positive mucosal CI was found in proximal AZ in 6 (43%) of 14 HD pull-through,
among which 1 case lacked submucosal nerve hypertrophy in the proximal AZ. All 14
HD pull-through cases showed an increasing trend/gradient of PC from AZ toward TZ.
Conclusion Based on our findings, the presence or absence of mucosal CI positivity and submucosal
nerve hypertrophy may not reliably diagnose vsHD in rectal suction biopsy. While we
acknowledge that the density of mucosal innervation in variable contexts and anatomical
locations is unknown and yet to be explored, our study suggests that an increasing
trend of positive mucosal CI from AZ toward TZ by IPA might prove to be a useful tool
for the diagnosis of vsHD in the future.
Keywords
image processing - Hirschsprung - very short - calretinin - analysis
